Biochimica et Biophysica Acta (BBA) - Bioenergetics

Mechanistic interpretation of photosynthetic energy-transfer models requires more than reproduction of experimental observables: a model intended to support mechanistic claims should also respond consistently when its proposed functional organizations are removed. Here, we evaluate whether a calibrated PSI-LHCI transport surrogate encodes physically meaningful principles by applying a 2x2 factorial knockout framework that independently removes site-energy heterogeneity and coupling-strength heterogeneity in a 155-pigment network. All perturbations were evaluated using the same Full-model calibration without parameter refitting. Although the calibrated model reproduced a high excitation-trapping yield, eliminating either energetic or coupling heterogeneity unexpectedly improved its apparent transport performance. A strict zero-coupling control confirmed that coupling itself remained necessary for network-mediated reaction-center access, whereas the supplied organization of coupling strengths was not supported by the surrogate. An audit of the model inputs further identified peripheral localization of all lowest-energy states and effective coupling scales far above those used in structure-based chlorophyll Hamiltonians. These findings do not imply that native PSI favors flat energy landscapes or uniform couplings. Instead, they show that endpoint agreement alone does not validate a mechanistic interpretation of a pigment-network model. Factorial knockout analysis provides a falsification-oriented framework for separating physical necessity from proposed organization, diagnosing numerical compensation, and identifying the constraints required for more predictive models of PSI-LHCI energy transfer.

Lysine acetylation is an evolutionarily conserved, post-translational modification that regulates metabolism and protein function, yet its role in archaeal electron transfer systems remains poorly understood. Here, we investigated lysine acetylation of the 2Fe-2S ferredoxin HvFdx (HVO_2995) and its flavin-dependent oxidoreductase HvFdR (HVO_2345) partner in the halophilic archaeon Haloferax volcanii. Genetic and biochemical analyses established HvFdx as an essential 2Fe-2S ferredoxin with a midpoint redox potential of -385 mV. Lysine acetylation of HvFdx was found to occur primarily at K119, a residue positioned near the [Fe-S] cluster interface, and to modulate electron transfer capacity without impacting Fe-S cluster incorporation, midpoint potential, or protein abundance. In contrast, HvFdR was found lysine acetylated at multiple sites in a manner consistent with a non-enzymatic mechanism that resulted in altered flavin binding, enzymatic activity, and thermal stability. Lysine acetylation of HvFdx was found to stimulate electron flow from HvFdR as measured by an anaerobic NADPH [->] HvFdR [->] HvFdx [->] DCIP assay. 3D structural modeling, proteomic, biochemical, and genetic assays suggest the haloarchaeal GNAT-family acetyltransferase homolog HVO_2874 as a candidate enzyme associated with HvFdx lysine acetylation and optimal growth of H. volcanii. Together, these findings demonstrate that lysine acetylation differentially regulates archaeal redox-active proteins and functions as an important mechanism coordinating redox metabolism in H. volcanii.

Members of the FAD-dependent oxidoreductase family (IPR050260) play diverse and key roles in maintaining cellular redox balance, yet the functions of many distinct subgroups within this family remain unknown. Here, we define the biochemical and physiological functions of the Haloferax volcanii flavin-dependent oxidoreductase HvFdR (HVO_2345; fdr), a haloarchaeal member of a previously uncharacterized IPR050260 subgroup. HvFdR binds FAD and catalyzes NAD(P)H oxidase, diaphorase and ferredoxin reductase activities, with a kinetic preference for NADPH over NADH and catalytic properties that are strongly influenced by oxygen availability. Under stoichiometric conditions, HvFdR mediates reverse electron transfer to NADP, suggesting that intracellular nicotinamide nucleotide pools regulate electron flow bidirectionally. Consistent with this reversibility, HvFdR bound-FAD exhibits a low midpoint redox potential (-413 mV), supporting its capacity to function as an electron donor. Deletion of fdr impairs growth and elevates intracellular NADPH levels, consistent with a role for HvFdR in maintaining NADP(H) homeostasis. Conserved residues K47 and Y323 are identified as determinants of HvFdR electron transfer activity and may function as a regulatory gate that modulates electron flow while limiting excessive H2O2 production under aerobic conditions. Together, these findings establish HvFdR as an oxygen-responsive flavin-dependent oxidoreductase that contributes to cellular redox homeostasis and provides functional insight into a previously uncharacterized subgroup of the IPR050260 family.

Nitrite can be reduced to nitric oxide (NO) by several heme- and molybdenum-containing proteins, including mitochondrial cytochrome c oxidase (Cco). This activity, designated Cco/NO, has been implicated in hypoxic signaling, but its regulation and quantitative significance relative to other NO-producing systems remain uncertain. We examined its modulation by adenine nucleotides using detergent-solubilized yeast and mouse brain mitochondria supplied with 1 mM nitrite and an ascorbate/TMPD/cytochrome c electron-donor system. ADP and ATP differentially modulated Cco/NO activity, and ADP extended measurable NO formation across the entire oxygen range tested, up to the assay ceiling of 175 {micro}M O2. Nucleotide regulation was also isoform-dependent: ATP slightly inhibited Va-containing Cco but strongly stimulated Vb-containing Cco under anoxic conditions. Rates normalized to cytochrome aa demonstrate multi-turnover nitrite-reductase capacity under these substrate-driven assay conditions. Both the cellular ADP/ATP ratio and subsequently assayed Cco/NO activity increased transiently following a hypoxic shift. These findings establish metabolic and isoform-dependent gating of the catalytic capacity of Cco/NO; they do not establish its fractional contribution to total cellular NO or its operation at physiological nitrite concentrations in intact, coupled mitochondria. This research was supported by CONICET Grant PIP 706 (research team member P.R.C.) and National Institutes of Health Grant GM30228 to R.O.P.

Photosymbioses, or associations between heterotrophs and photoautotrophs, are widespread and indispensable in today's ecosystems. The chloroplasts of algae and land plants, which are at the heart of most of earth's primary production, stem from ancient photosymbioses. Photosymbioses often combine heterotrophy and autotrophy and must thus efficiently allocate resources between these two costly cellular processes. We currently lack a clear picture of how photosymbioses allocate their valuable cellular resources in response to environmental change. In this study, we combine growth assays, automated fluorescence microscopy, transmission electron microscopy, and mass spectrometry-based proteomics to explore the physiology and cellular resource allocation of the ciliate-green alga photosymbiosis of Paramecium bursaria. In nutrient-rich environments that resemble P. bursaria's natural habitat, the maximum growth rate attained saturates regardless of light intensity. The green algae thus do not provide a benefit in nutrient-replete conditions, and the photosymbiosis primarily functions heterotrophically. The green algae occupy a remarkably similar and constant volume fractions across contrasting light environments despite displaying clear photo-physiological adaptation. This is true regardless of a clear physiological cost of the photosymbionts; aposymbiotic hosts always display higher growth rates in the dark. The host does not decrease 'symbiont load' in environments where green algae are not beneficial. Moreover, in the dark, the green algae are fully dependent on their hosts and take up a larger proteome mass fraction that increases with prey abundance. Differential protein expression analyses suggest that acetate and amino acids are the preferred sources of carbon and nitrogen for the green algae in the dark. The stable persistence and higher resource uptake by the photosymbionts in the dark argue against a view where hosts have full control over and selfishly exploit their symbionts.

Ferredoxin-NADP+ reductases (FNRs) are ubiquitous flavoenzymes that catalyse the reversible transfer of electrons between iron-sulfur ferredoxins and the pyridine nucleotide pool, thereby occupying a central position in diverse redox metabolic pathways including photosynthesis, nitrogen fixation, and detoxification of reactive oxygen species. Although FNR activity was demonstrated in cell extracts of Clostridium pasteurianum more than five decades ago, the gene encoding this activity has remained unidentified. In the present study, a systematic bioinformatic screen of all 3,797 predicted proteins from the C. pasteurianum genome was conducted using conserved FAD- and NAD(P)+-interacting residues from structurally characterised reductases as search templates. This analysis identified a single candidate, AQ984_05830, which is annotated as a sporulation protein but possesses all six predicted cofactor-interacting residues. Heterologous expression and cytochrome c reduction assays confirmed ferredoxin-dependent reductase activity, with a wild-type kcat of 0.007 min-1--a value orders of magnitude lower than those reported for canonical FNRs. A parallel genome-wide screen further revealed a repertoire of ferredoxin-like carriers, suggesting that C. pasteurianum distributes hydrogen-derived electrons among multiple ferredoxins to serve diverse metabolic fates, of which NADP reduction by CpFNR is one. Alanine scanning mutagenesis of five predicted cofactor-interacting residues revealed that K68A and K73A mutations abolished activity, whereas T64A, T185A and S202A mutations improved catalytic efficiency (kcat/Km) for NADH by 14 to 18 folds. AlphaFold structure prediction combined with SwissDock and ClusPro molecular docking simulations placed the FAD binding site centrally between the NAD(P)H and ferredoxin binding domains, consistent with the expected electron relay architecture. Structural analysis of the beneficial mutations suggests that disruption of hydrogen bonds flanking a flexible coil (residues 186-199) propagates conformational effects to the NAD(P)H binding loops, rationalising the improved substrate affinities. These findings expand the known functional diversity of the FNR superfamily and suggest an unrecognised role for redox regulation during endospore formation in C. pasteurianum.

Cardiolipin (CL) is a phospholipid found in the inner mitochondrial membrane (IMM) where it increases the efficiency of ATP regeneration. We have investigated the hypothesis that this increase may result in part from CL concentrating H+ at the IMM surface through electrostatic interactions as the CL polar head is a dianion at physiological pH. To this aim, we compared the concentrations and movements of H+ at the surface of giant planar phosphatidylcholine (PC) membranes and 20% CL enriched PC membranes by recording their surface pH with the membrane-grafted pH probe fluorescein DHPE. CL enrichment of the membranes increased their surface H+ activity by a ~4 factor. Moreover, we observed non-gaussian spatial H+ concentration profiles with distance from a point H+ source with both PC and CL membranes suggesting that both lipids also induce interactions between probe molecules. A whole bath pH variation revealed that these interactions allow the traveling of reversible acidification fronts with constant speed over the membrane between high and low pH states. A reaction-diffusion model of these observations suggests that membranes support these fronts through a mechanism of autocatalytic (de)protonation of the membrane surface. In mitochondria, these fronts would result in transitions between high and low pH states, the low one having a larger H+ concentration in CL-enriched regions of the IMM. Such an increase at the inner leaflet of the IMM may increase efficiency of the respiratory chain whereas the increase at the outer leaflet may boost the ATP synthase rate.

Ferritins are ubiquitous iron homeostasis proteins found across the tree of life that form conserved 24-subunit cages with octahedral (4-3-2) symmetry. New types of ferritins and ferritin-like proteins are being continuously discovered, such as mini-bacterioferritins, which form smaller shells of 12 subunits, and double-ferritin-fold proteins, which act as ferroxidases but do not form shells. Here, we describe double-ferritin-fold proteins from Asgard archaea, dubbed dFTNs, and determine Cryo-EM structure of a representative from Candidatus Heimdallarchaeum endolithica. The protein forms a dodecameric shell with tetrahedral (2-3) symmetry. N-terminal (NTD) and C-terminal (CTD) domains are bridged by an ordered linker and are related by two-fold rotational pseudosymmetry. C-terminal -helix (helix E) that forms the four-fold channel in classic ferritins is repositioned to be the helix 2 out of 5 ferritin domain -helices in dFTN, with two such helices from NTD and two helices from CTD forming a pseudo-four-fold symmetry structural element. Four three-fold channels are formed by NTDs, and four other such channels are formed by CTDs. The overall arrangement of dFTN ferritin domains is similar to that of protomers in classic ferritin shells. Altogether, our findings expand the range of known ferritin family proteins and provide insight into Asgard archaea iron metabolism.

Mitochondria integrate bioenergetic metabolism, redox control, genome maintenance, and stress signaling across all eukaryotes. Although plant and human mitochondria diverged substantially during evolution, both systems retain systems-level principles for sensing mitochondrial dysfunction and communicating stress signals to the nucleus. Here, we develop an integrative comparative in silico framework to evaluate whether plant mitochondrial stress signaling can provide a useful conceptual model for interpreting human mitochondrial disease vulnerability. Core Arabidopsis thaliana regulators representing alternative respiration, mitochondrial retrograde signaling, translational stress control, and genome surveillance were compared with functionally analogous human regulators involved in integrated stress response (ISR) signaling, mitochondrial DNA maintenance, and mitochondrial disease phenotypes. Domain architecture, protein-protein interaction topology, enrichment profiles, disease-gene associations, and promoter motif architecture were integrated to assess cross-kingdom convergence at the level of stress-response organization rather than direct orthologs. The plant network formed a compact AOX-NAC-centered stress module associated with respiratory flexibility and retrograde signaling, whereas the human network displayed expanded ISR and mtDNA maintenance modules enriched for mitochondrial disease associations. Promoter motif analyses further indicated lineage-specific transcription factor signatures but broadly comparable stress-responsive regulatory logic. Collectively, these results support the concept that plant mitochondrial stress systems represent simplified resilience-oriented architectures that can help generate experimentally testable hypotheses about failure points in human mitochondrial stress responses.

Photoprotection against excess energy is essential for the survival of photosynthetic organisms under adverse conditions. In plants, excess energy can be dissipated as heat through non-photochemical quenching (NPQ) of chlorophyll fluorescence, involving the trimeric light-harvesting complex II (LHCII), the major antenna of photosystem II. How NPQ affects antenna proteins remains debated, especially as most studies focus on short-lived components artificially induced in vitro. Here, we characterize the effects of qH, a long-lived NPQ component, on the fluorescence properties of natively quenched LHCII. Single-molecule fluorescence measurements, combined with biochemical and biophysical ensemble approaches, reveal a larger and more quenched subpopulation of LHCII trimers exhibiting fluorescence intermittency in samples with qH compared to those without. This behavior is linked to a small conformational change that stabilizes a quenched state, enhancing photoprotection at the antenna level. These findings provide new insights into sustained NPQ and its role in regulating energy dissipation under natural light conditions.

Within a single human lifetime, the spectral environment has been fundamentally reshaped. Broadband daylight, rich in infrared (IR) photons arising from solar and atmospheric physics, has been replaced in the built environment by narrow, engineered spectra that largely exclude long wavelengths. While modern lighting is optimised for vision, the non-visual photobiology of metabolism may depend on spectral components that are now absent. When expressed in photon-energy units, the solar spectrum exhibits a broad maximum near 0.75 eV. This range overlaps with the activation and reorganisation energies governing mitochondrial electron-transfer kinetics. Within a Marcus-type framework, IR photons are therefore positioned to modulate rate-limiting metabolic steps by biasing barrier-crossing probabilities rather than supplying chemical energy. These wavelengths also penetrate deeply into tissue in a scattering-dominated regime, forming a diffuse internal photon field capable of interacting with distributed mitochondrial networks. We propose the term photometabolism: a solar-driven, non-photosynthetic modulation of core metabolic processes. A scaling analysis shows that photon interception in this band varies with body mass in parallel with basal metabolic rate, suggesting that ambient sunlight provides sufficient flux to influence metabolic kinetics across the biosphere. These findings have implications for physiology, ecology and the design of indoor environments whose lighting spectra increasingly diverge from their evolutionary context.

Photosynthetic electron transfer relies on the coordinated function and spatial organization of large protein complexes within the thylakoid membrane. The cytochrome b6f complex (b6f) functionally interconnects photosystem (PS) II and PSI in photosynthetic electron transfer and is equally distributed between appressed and non-appressed thylakoid membranes. Here, we investigate the functional link between the lateral distribution of b6f and efficient photosynthetic electron flow in Chlamydomonas reinhardtii. We engineered strains with stromal fusions between PetA of b6f and two fluorescent proteins (FPs) of different molecular mass: Clover and ATeam. Under oxic conditions, these strains exhibited significantly slower electron transfer rates (ETR), lower PSII quantum yields, and increased donor-side limitation of PSI. State transitions were diminished in the fusion strains, accompanied by a strong impairment of STT7-dependent function, suggesting that the presence of fused FPs at b6f interfere with STT7 function. Yet, ETR phenotypes were STT7-independent and FP fusion did not impact intrinsic b6f function. In situ cryogenic electron tomography revealed a significant depletion of b6f from appressed thylakoid membranes in the ATeam strains, while the overall membrane protein concentration remained unchanged. Overall, our data indicate that a balanced distribution of b6f between appressed and non-appressed thylakoid membranes is essential for regulating photosynthetic electron transfer, highlighting the functional importance of thylakoid molecular architecture in vivo.

Soil microalgae endure harsh terrestrial stressors, such as intense light. Eustigmatophytes are an independent evolutionary branch within stramenopiles and occupy diverse aquatic and terrestrial environments, but the structural organization of their photosynthetic apparatus remains poorly understood. Here, we determined the cryo-electron microscopy structure of a photosystem I-light-harvesting complex I (PSI-LHCI) supercomplex bound with ferredoxin-NADP+ oxidoreductase (FNR) from the terrestrial eustigmatophyte Vischeria stellata at 2.44 [A] resolution. The supercomplex contains a monomeric PSI core associated with only three LHCI subunits, representing the smallest PSI-LHCI reported among structurally characterized red-lineage PSI complexes composed of violaxanthin-Chl a proteins (VCPs). The three VCPIs with distinct structure features and arrangements form a compact belt along the PsaL-PsaI-PsaM side of PSI. The structure also resolves a 43-residue N-terminal segment of FNR (FNR-N) bound to the PSI stromal surface, which is stabilized by both a eustigmatophyte-conserved insertion in PsaL and the N-terminal region of PsaD. In contrast, the catalytic region of FNR was not resolved, suggesting conformational flexibility. Computational simulations indicate potential excitation-energy-transfer pathways connecting the three VCPI subunits to the PSI core and highlight lineage-specific pigments that maintain energetic connectivity within the exceptionally compact antenna. Our analysis further reveals conservation of FNR tethering despite pronounced diversification of antenna size and organization. These findings uncover a modular evolutionary principle in which PSI acceptor-side organization is retained while the light-harvesting antenna is extensively remodeled, providing a framework for understanding the diversification of photosynthetic energy conversion across ecological transitions.

Motivation: Continuing advances in genome and metagenome sequencing expand the number of identified conserved protein families that remain functionally uncharacterized and contain domains of unknown function (DUFs). Functional-association resources such as STRING provide biological context, but mostly do not distinguish indirect association from physical interaction. We assessed whether AlphaFold 3 complex prediction, combined with STRING evidence and domain-level analysis of interfaces and interaction partners, can help identify and characterize DUF-containing proteins. Results: We generated four structural-prediction cohorts from STRING associations involving DUF-containing proteins and evaluated the predicted complexes using interface ipSAE, average pLDDT and buried surface area. An L2-regularized logistic regression model was trained on an initial cohort of predictions from high-confidence STRING associations to prioritize DUF-containing candidates likely to produce structurally confident AlphaFold 3 complexes. The model was then applied across all 12,535 organisms represented in STRING v12.0, followed by grouping into DUF-family and partner-architecture modules, covering 2,076 unique DUF families. The final L2-model screen contained 12,298 successfully modelled protein pairs, including 1,208 (9.82%) complexes meeting a strict-confidence criterion and 2,433 (19.78%) meeting a more liberal confidence criterion. Two examples suggest roles for DUF4130 in nucleic-acid-associated radical-SAM biology and DUF5819 in a bacterial system related to vitamin-K-dependent carboxylation. Availability and implementation: Predicted structures and associated metadata are available through Zenodo at https://doi.org/10.5281/zenodo.21875362. The model implementation and code used to generate the analyses and figures are available at https://github.com/linoriep/Proteome-scale-structure-prediction-of-DUF-containing-protein-protein-interactions.

Elephant seals are remarkable breath-hold divers, capable of remaining submerged for up to two hours during diving bouts. These dives entail repeated, extreme hypoxia/reoxygenation events that would induce severe lipid peroxidation and tissue dysfunction in most mammals. Here, we show that primary vascular endothelial cells derived from elephant seals possess an intrinsic resistance to lipid peroxidation. Comparative transcriptomic and lipidomic profiling across seal, human, and sheep cells identified ferroptosis - an iron-dependent, lipid peroxidation-driven cell death pathway - as uniquely regulated in seal cells following hydroperoxide exposure. Mechanistically, seal cells exhibit robust baseline expression of acyl-CoA synthetase long-chain family member 3 (ACSL3), alongside rapid, seal-specific induction of the sole mammalian iron exporter, ferroportin (SLC40A1). Functional validation using genetic and pharmacological approaches revealed that seal cells are naturally enriched in monounsaturated fatty acids and triglycerides and utilize lipid droplet biogenesis and active iron export as dual protective axes to evade lipid peroxidation. Together, these findings show that elephant seal cells employ a coordinated cytoprotective network of lipid remodeling and iron handling to withstand the severe challenges of deep diving. SIGNIFICANCE STATEMENTDeep-diving marine mammals repeatedly experience extreme hypoxia-reoxygenation events that would induce severe oxidative damage in most terrestrial mammals. However, vascular cells derived from seals naturally resist lipid peroxidation, a major driver of ischemia-reperfusion injury. Here, we show that elephant seal endothelial cells evade lipid peroxidation through two complementary mechanisms: lipid droplets that sequester peroxidation-prone phospholipids, and rapid iron export that limits lipid peroxide formation. These findings reveal naturally evolved cellular strategies that protect against vascular oxidative stress, offering new insights into physiological resilience against ischemia-reperfusion injury.

RationaleRecent viral metagenomic studies have identified a plethora of enzyme-encoding genes in Phycodnaviridae viruses that are not strictly required for viral replication. These enzymes hold an unexpected metabolic potential during the infection process with their specific green algae host. As neither their role in the infection process nor the subcellular localization of these proteins has been experimentally characterized, comparative sequences, structural and biochemical in silico analyses can help generate functional and localization hypotheses. MethodsIn a recent viral metagenomic dataset, we identified a collection of viral homologs involved in bilin biosynthesis: heme oxygenase (vHMOX1) and Phycocyanobilin:Ferredoxin oxidoreductase (vPcyA). Viral and algal homologues were compared through sequence analyses and AlphaFold3 structural predictions. Predicted biochemical properties were analyzed for their compatibility with subcellular compartments. Active site architecture and putative substrate binding were compared between viral and algal proteins using AlphaFold3 and experimentally resolved structures. ResultsViral HMOX1 and PcyA sequences are truncated compared to algal homologs, lacking the N-terminal extension associated with chloroplast targeting. However biochemical properties, including isoelectric point and surface charge distribution, are compatible with localization in chloroplast stroma. Structural comparisons reveal modifications in the viral HMOX1 active site, including partial substrate reorientation and substitutions of key residues, consistent with modified heme-binding properties. In contrast, vPcyA models show no significant differences to their algal counterparts. ConclusionsActive site remodeling in vHMOX1 protein models suggests that these viral homologues may have evolved distinct heme-binding properties. Unlike vPcyA, vHMOX1 homologs appear to have diverged more substantially from their algal counterparts, potentially reflecting functional specialization in the viral infection context. One sentence summary of key findingsOur bioinformatic analyses expand the repertoire of auxiliary metabolic genes in Phycodnaviridae by identifying a conserved heme degradation pathway, non-canonical vHMOX1/PcyA targeting and structural rearrangements surrounding the catalytic sites of viral HMOX1.

Photosystem II (PSII) is in eukaryotic phototrophs is generally considered to operate within a more restricted spectral range than photosystem I (PSI), in which long-wavelength chlorophylls are a well-established feature of the peripheral antenna. Whether eukaryotic PSII can acquire comparable far-red-associated properties through lineage-specific antenna diversification has remained unclear. Here we present a 3.09 [A] cryo-electron microscopy structure of the C2S2M2L2 PSII supercomplex from Euglena gracilis, a euglenophyte species harbouring a secondary plastid and unusual light-harvesting system. We show that the euglenophyte-specific antenna protein LhcE9 occupies the position corresponding to canonical Lhcb5, but in a markedly different orientation that creates a distinct interface with the PSII core, particularly with CP43. Combined structural, spectroscopic, mutagenesis and proteomic analyses support LhcE9 as the stably bound PSII antenna subunit most closely associated with the far-red state in the supercomplex. Excitation-energy-transfer calculations further indicate two fast lineage-specific antenna-to-core routes mediated by LhcE9 and PsbX. Together, these findings reveal an unexpected mode of PSII antenna diversification and provide a structural framework for far-red-associated light harvesting in PSII.

Toxicological omics studies require comprehensive metadata to support reproducibility, interoperability, and regulatory reuse. However, metadata requirements differ across public repositories, reporting frameworks, and laboratory workflows, resulting in inconsistent annotation and limited data integration. To address this challenge, we developed MeSyTo (Metadata for Systems Toxicology), an ontology-driven framework for harmonizing metadata across toxicological omics. Metadata concepts from public repositories, the OECD Omics Reporting Framework (OORF), community standards, and institutional workflows were semantically aligned and implemented as the MeSyTo Metadata Model (MMM). The MMM serves as the basis for the automatic generation of SHACL validation shapes and framework-specific metadata profiles, while curated value sets are represented as SKOS controlled vocabularies to support metadata collection and validation. The current implementation comprises 105 ontology classes and 527 data properties and supports transcriptomics, proteomics, and metabolomics. A prototype web application demonstrates ontology-driven metadata collection with integrated semantic validation and ontology-based term resolution. The ontology, validation shapes, controlled vocabularies, generation scripts, and software are publicly available as open-source resources. MeSyTo provides a reusable semantic foundation for harmonized, machine-actionable metadata and facilitates repository submission, regulatory reporting, and interoperable data exchange across toxicological omics studies.

The navigational prowess of migratory birds is thought to arise from light-dependent radical-pair chemistry in cryptochrome 4 (CRY4), yet the slow structural transitions that couple photochemistry to signaling remain elusive. Here, we combine temperature-controlled steady-state UV-visible spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to elucidate the photochemical and conformational dynamics of pigeon CRY4 (ClCRY4). Steady-state measurements at 5-25 {degrees}C reveal that lower temperatures slow FAD photoreduction and prolong the FAD neutral semiquinone signaling state. This occurs without a solvent kinetic isotope effect, implicating a conformational change rather than proton transfer as the rate determining step in FAD neutral semiquinone formation. Simultaneous HDX-MS under blue-light exposure identifies protection near the FAD-binding site and C-terminal region. To enhance sensitivity, we developed a pump-probe HDX-MS approach at 10 {degrees}C. This reveals eight peptides (within the phosphate-binding loop, protrusion motif, electron-transfer-chain loops and C-terminal tail) that exhibit rapid ([≤]10 s) and sustained light-induced protection, delineating early conformational rearrangements as a prerequisite for FAD neutral semiquinone accumulation. The findings of slower onset HDX protection as well as a bimodal pattern of deuterium uptake in the phosphate-binding loop further identify a local redistribution of conformational substates on the time scale of the accumulation of the signaling species. Site specific mutagenesis within the CTT supports the findings, which lead to a model in which blue light triggers rapid clamping down of protein near the two regions of spin pair separation, followed by a rate limiting closure of a surface loop. The resolution of time-dependent structural transitions that follow photoactivation of CRY4 resolves the interface between quantum radical-pair formation and classical conformational changes, while providing an enhanced structural framework for the molecular events that underlie avian magnetoreception.

Chlorophyll (Chl) is essential for oxygenic photosynthesis, binding to membrane proteins for light harvesting and electron transfer. In angiosperms, Chl synthesis is halted in darkness, preventing accumulation of Chl-binding photosynthetic complexes in etioplasts. However, etioplasts assemble a dimeric Cytochrome b6f (Cyt b6f) complex, uniquely binding protochlorophyll (Pchl), the esterified derivative of protochlorophyllide (Pchlide). This indicates an evolutionarily conserved structural or functional role for Pchl distinct from the Chl bound in Cyt b6f in chloroplasts. Here we show that upon light-induced Chl synthesis in-vivo and in-vitro, Chl accumulation in Cyt b6f dimers precedes photosystems I and II. We find that chlorophyllide and Chl bind to the light-harvesting-like protein 3 (LIL3), supporting a role for LIL3 in early Chl allocation that extends its described role in stabilizing geranylgeranyl reductase. We determine a dissociation constant of 246.6 {+/-} 37 nM for Chlide binding to LIL3 in-vitro and show that Cyt b6f monomers and LIL3 co-migrate with Chlide in native PAGE, whereas Cyt b6f dimers and LIL3 co-migrate with Chl. These results indicate that Chlide binding to LIL3 chaperones esterification to Chl and reduction of geranylgeraniol, and that Chl release with Cyt b6f dimerization prioritizes Chl binding to Cyt b6f assembly during de-etiolation.